Project Abstract Management of Chronic Myelogenous Leukemia (CML) has improved greatly since the advent of oral tyrosine kinase inhibitors (TKIs). However, as the use of these agents has been adopted widely, it has become increasingly clear that different polymorphisms of ABL1 kinase influence the effectiveness of the TKI. One mutation in ABL1 kinase is the T315I positive CML that is resistant to most tyrosine TKIs. Ponatinib (Iclusig, Ariad Pharmaceuticals) uniquely targets the T315I mutation in the BCR-ABL protein. This TKI has a wider range of kinase targets than other agents and has been effective in salvage therapy in patients who have failed other treatments. However, it is not without consequences. In the PACE trial, data indicate that when the sum of cardiovascular (CV) events (myocardial infarction, stroke, limb ischemia, coronary artery stenosis, and occlusions of brain and peripheral circulation) were obtained in the ponatinib- treated patients, ~29% of patients had a negative outcome. It was not clear from the clinical trial what were the conditions in these patients that were associated with the negative CV outcomes. We created a murine model to determine if ponatinib treatment is distinct from imatinib or nilotinib in terms of increasing thrombosis risk. In mice treated with the various TKIs under steady-state conditions, ponatinib, unlike imatinib or nilotinib, increased risk for arterial thrombosis. Further we observed that ponatinib-treated mice have increased reactive oxygen species and apoptosis in vessel wall and hyper- responsive platelets ? two features that contribute to heightened clinical thrombosis. These effects in vivo are mechanistically related. Treatment of mice with pioglitazone is able to correct vessel wall injury, correct platelet hyperactivity, and correct arterial thrombosis times to normal. The overall hypothesis that guides this application is that ponatinib's pharmacologic inhibition of vascular ABL1 kinase and platelet p-Lyn results in reduced antithrombotic vascular function and hyperactive platelets, leading to heightened arterial thrombosis. Our data indicate that ponatinib increases risk for thrombosis by blocking vasculo-protective mechanisms and activating platelets. The specific aims of this proposal are: 1) Characterize the mechanism(s) by which ponatinib reduces the antithrombotic nature of the vascular wall; 2) Characterize the mechanism(s) by which ponatinib treatment creates platelet hyperactivity and 3) Determine what classes of agents have potential to nullify the negative effects of ponatinib. These investigations will determine the mechanism(s) for ponatinib-associated cardiovascular events. They will provide a mechanistic basis for recognition of therapies to protect patients from the risk of increased adverse cardiovascular events while receiving ponatinib treatment. We will demonstrate how to ameliorate the negative cardiovascular effects of ponatinib with conjoint use of certain medications. This research will provide a means for continued safe use of ponatinib in CML patients with cardiovascular risk factors with the T315I mutation. Since patients with the T315I gatekeeper mutation in BCR-ABL protein are the most difficult to treat, our investigations should provide for a means to protect them from risk from adverse cardiovascular complications with ponatinib treatment.